1. Field
The present invention relates to a composition comprising β-hydroxy-β-methylbutyrate (HMB) and methods of using HMB to treat, prevent or improve diseases or conditions that can benefit from autophagy enhancement and/or modulation.
2. Background
The word autophagy is derived from Greek words that translate into “self-eating”. It is a physiologic process that is geared towards recycling of cellular components after destruction of cells in the body. It maintains homeostasis or normal functioning during basal condition, but more importantly during periods of cellular stress. In this regard, autophagy provides an alternate source of intracellular building blocks and substrates that may generate energy to enable continuous cell survival. This process is seen in all eukaryotic systems including fungi, plants, slime mold, nematodes, fruit flies and insects, rodents (laboratory mice and rats), humans.
Autophagy is an essential cellular multistep degradation pathway that terminates in the lysosomes. Autophagy involves the formation of double membrane bound vesicles (autophagosomes) that sequester cytoplasmic cargo consisting of aggregated proteins, defective organelles or lipid droplets for delivery to lysosomes. The process is important for cellular health and for cell survival during stress. Through its function in degrading cytoplasmic protein aggregates and defective organelles, autophagy contributes to maintaining quality control of cellular proteins and organelle function. There is a basal rate of autophagy in most cells that serves to maintain protein homeostasis i.e. “proteostasis” and organelle function and during stress, upregulation of autophagy corrects stress-induced cellular changes to restore proteostasis.
In addition to its housekeeping functions, degradation of protein and lipid via autophagy provides cellular energy. Autophagy is induced by energy challenges such as calorie restriction, fasting or exercise. A specific form of autophagy, called lipophagy, mobilizes lipid stores using autophagosome. The lipids, hydrolyzed by lysosomal acid lipases, are used for energy through beta-oxidation. Autophagic lipid catabolism (lipophagy) is a non-classical mechanism by which cells mobilize lipid stores and maintain cellular homeostasis. Defective lipophagy has been linked to metabolic diseases, such as fatty liver disease, obesity and atherosclerosis. Exercise potently induces autophagy in muscle and heart where it mediates endurance and the beneficial effects of exercise on muscle glucose homeostasis.
Basal autophagy and its upregulation in response to stress are essential for maintenance of tissue health in the muscle, liver, brain, heart, intestine, pancreas, and adipose tissue etc. For example blocking autophagy in the liver by blocking ATG7 or ATG5 results in endoplasmic reticulum stress and induces insulin resistance. Similarly, blocking autophagy by deletion or suppression of the key autophagy protein ATG7 or its downstream mediator ATG5 leads to neuronal death and neurological abnormalities. Autophagy deficits occur with aging, which is often characterized by loss of protein quality control and accumulation of intracellular damage. In part this damage might reflect an age-related decrease in AMPK signaling which reduces autophagy. Autophagy modulates aging through an effect on lipid homeostasis. Enhancing autophagy activity, via overexpression of key autophagy genes or by calorie restriction, promotes anti-aging phenotypes and increases lifespan (20%) in mice.
There is strong evidence for transcriptional and epigenetic control of autophagy. (Pietrocola F., Regulation of Autophagy by Stress-Responsive Transcription Factors. Semin. Cancer Biol. 2013; 23:310-322). In mammals, transcription factor EB (TFEB) and members of the forkhead box protein class O (FOXOs) are master transcriptional regulators of autophagy.
BNIP3 is a transcriptional target of FOXO1 and inhibits the mTOR signaling pathway independent of AMPK. TFEB is transcriptionally induced by CREB activation and is a major transcriptional regulator of autophagy linked genes and also of lysosomal genes.
TFEB appears to exert the most global control over autophagy by regulating multiple steps of autophagy, such as autophagosome biogenesis, substrate targeting, and lysosome degradation by managing expression levels of several autophagy and lysosomal genes. (Settembre C. TFEB Links Autophagy to Lysosomal Biogenesis. Science 2011; 332; 1429-1433). TFEB regulates the autophagic flux by coordinating the expression of genes with functions at all stages of the autophagy process. Further, TFEB regulates lipophagy in adipocytes. (Kaur J, Debnath J. Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol. 2015; 16(8):461-72).
TFEB activity can be impaired by the age-related accumulation and aggregation of disordered proteins in a variety of metabolic and lysosomal storage diseases. Disruption of normal autophagic processes contributes to the development of metabolic disorders, such as obesity and insulin resistance. Defective lipophagy underlies the basis for the metabolic syndrome of aging.
Autophagy deficits are observed early in Alzheimer's, amyotropic lateral sclerosis (ALS), neurodegeneration, cardiomyopathy, and osteoarthritis. Defective autophagy may also contribute to obesity-associated insulin resistance and autophagy upregulation (example by calorie restriction) improves insulin sensitivity. Autophagy dysfunction is also a major contributor to diseases such as neurodegeneration, liver disease such as hepatitis, hepatic steatosis, cirrhosis, nonalcoholic steatohepatitis (NASH), and fatty liver disease, cancer, autoimmune disorders, metabolic disorders such as diabetes and/or insulin resistance, sarcopenia, and cardiovascular disorders such as stroke, cardiac atrophy, heart attacks, cardiomyopathy, and transient ischemic attacks. Autophagy is defective in humans with muscular dystrophy and studies have shown that this defect contributes to the pathogenesis of the disease. Increased autophagy can be induced by stresses such as nutrient starvation, infection and aging.
Autophagy can be considered somewhat of a double-edged sword; it can act as a tumor suppressor by preventing oncogenic protein substrates, toxic misfolded proteins and damaged organelles that initiate cancer, yet higher autophagic activity has been detected in tumor cells that may assist in the survival and growth of the tumors. Thus, autophagy modulators that act to normalize autophagy can act to prevent too little or too much autophagy and help regulate normal cellular function.
Autophagy is initiated by formation of double or multi-membrane vesicles in the cytoplasm forming autophagosomes, which engulf portions of the cytoplasm and organelles. The outer membrane of the autophagosome then fuses with lysosomes, and the inner membrane and the cargo are degraded by hydrolytic enzymes in lysosomes and then its constituents are recycled.
ATG (autophagy-related) proteins have been identified, and are essential for autophagosome biogenesis and maturation. Many of them gather at a double stranded cellular membrane. There are over 31 ATG proteins identified, including two essential ubiquitin-like conjugation systems that control autophagosome biogenesis. The first system results in the covalent attachment of ATG12-ATG5 proteins. In the second system, LC3 protein is conjugated to a lipid molecule, generally to a phosphatidylethanolamine (PE). Both conjugation systems require the function of E1-(ATG7) and E2-like enzymes (ATG3 and ATG10). ATG12-ATG5 stable complex eventually forms a larger complex with ATG16, resulting in the formation of an E3-like enzyme complex that catalyzes LC3-PE conjugation. ATG7 has been implicated in nutrient-mediated autophagy.
Failure of the autophagy process is a contributing factor to muscle disuse atrophy by failing to remove damaged mitochondria. The decrease in mitochondria turnover leads to an accumulation of dysfunctional organs and ensuing muscle damage. Impaired autophagy manifests as muscle atrophy, weakness, and degeneration of muscle myofibers. Autophagy has been shown to be a process that limits muscle damage.
Enhancing autophagy is a new target in developing treatments and prophylactic measures for cardiovascular disease, non-alcoholic fatty liver and insulin resistance. In contrast inhibiting autophagy might be beneficial in cancer by decreasing tumor cell survival to chemotherapeutic agents.
Further, levels of autophagy have been shown to diminish with age, resulting in increased muscle atrophy, decreased strength, decreased muscle function, and sarcopenia. Preserving or modulating the process of autophagy can improve these conditions that result from diminished levels of autophagy.
Aging results in the accumulation of various forms of molecular damage, as seen by malfunctioning organelles, defective enzymes, proteinaceous aggregates and/or DNA mutations. The incidence of chronic diseases, such as neurodegeneration, type II diabetes or cancer rises with age concomitantly with accumulating cellular damage. Normalizing or modulating autophagy acts to attenuate or avoid these age associated processes, thus prolonging an individual's “health span” and potentially their life span. Autophagy and/or lipophagy modulators increase the length of time an individual can lead an active lifestyle without suffering from conditions associated with aging, such as dementia, painful or reduced movement of limbs, diabetes, liver disorders, and/or cardiovascular disorders. These modulators can also decrease the time an individual suffers from infection.
Initial studies suggest that autophagy is regarded as a catabolic process, but recent studies have emphasized the importance of this pathway in sustaining and enabling anabolic pathways. It is now well-established that autophagy-derived nutrients produced from the catabolic degradation all support diverse biosynthetic pathways under basal conditions as well as during starvation. Thus, autophagy is a selective process. It has yet to be determined how autophagy is capable of mobilizing various nutrient pools towards specific anabolic functions. The present invention demonstrates that HMB acts a modulator of basal autophagy as all as autophagy during periods of nutrient excess such as obesity and insulin resistance.
Together, these studies described herein demonstrate a new mode of regulation of autophagy via FoxO3-dependent transcription and also that the autophagic/lysosomal pathway in muscle is regulated coordinately with the proteasomal pathway.
Nutrient starvation, inactivity or denervation, old age, and many diseases including cancer, diabetes, sepsis, renal failure, etc. are all associated with significant muscle loss (atrophy), loss of strength and endurance. The rapid muscle loss is attributed to accelerated protein breakdown secondary to activation of the ubiquitin-proteosomal pathways in the atrophying muscle. Foxo3, a member of the Forkead family of transcription factors, is highly activated under these conditions, and has been implicated as causing the muscle atrophy via transcription of a set of atrophy-related genes (“atrogenes”) including critical ubiquitin ligases, as well as stimulating autophagy suggesting the existence of a coordinated regulation of proteasomal and lysosomal systems.
Similarly, during starvation, autophagy selectively degrades lipid droplets, via the process of lipophagy, and this mediated by the expression of TFEB, of which activates the transcription of autophagy and lysosomal biogenesis genes. This factor coordinates the expression of lysosomal hydrolases, membrane proteins and genes involved in autophagy. In the presence of nutrients (and nutrient excess), the activity of these transcriptional factors are modulated by several other proteins, most important of which are mTOR proteins, involving coordinating the regulation of autophagy and lysosomal biogenesis.
HMB
Alpha-ketoisocaproate (KIC) is the first major and active metabolite of leucine. A minor product of KIC metabolism is β-hydroxy-β-methylbutyrate (HMB). HMB has been found to be useful within the context of a variety of applications. Specifically, in U.S. Pat. No. 5,360,613 (Nissen), HMB is described as useful for reducing blood levels of total cholesterol and low-density lipoprotein cholesterol. In U.S. Pat. No. 5,348,979 (Nissen et al.), HMB is described as useful for promoting nitrogen retention in humans. U.S. Pat. No. 5,028,440 (Nissen) discusses the usefulness of HMB to increase lean tissue development in animals. Also, in U.S. Pat. No. 4,992,470 (Nissen), HMB is described as effective in enhancing the immune response of mammals. U.S. Pat. No. 6,031,000 (Nissen et al.) describes use of HMB and at least one amino acid to treat disease-associated wasting.
The use of HMB to suppress proteolysis originates from the observations that leucine has protein-sparing characteristics. The essential amino acid leucine can either be used for protein synthesis or transaminated to the α-ketoacid (α-ketoisocaproate, KIC). In one pathway, KIC can be oxidized to HMB and this account for approximately 5% of leucine oxidation. HMB is superior to leucine in enhancing muscle mass and strength. The optimal effects of HMB can be achieved at 3.0 grams per day when given as calcium salt of HMB, or 0.038 g/kg of body weight per day, while those of leucine require over 30.0 grams per day.
Once produced or ingested, HMB appears to have two fates. The first fate is simple excretion in urine. After HMB is fed, urine concentrations increase, resulting in an approximate 20-50% loss of HMB to urine. Another fate relates to the activation of HMB to HMB-CoA. Once converted to HMB-CoA, further metabolism may occur, either dehydration of HMB-CoA to MC-CoA, or a direct conversion of HMB-CoA to HMG-CoA, which provides substrates for intracellular cholesterol synthesis. Several studies have shown that HMB is incorporated into the cholesterol synthetic pathway and could be a source for new cell membranes that are used for the regeneration of damaged cell membranes. Human studies have shown that muscle damage following intense exercise, measured by elevated plasma CPK (creatine phosphokinase), is reduced with HMB supplementation within the first 48 hrs. The protective effect of HMB lasts up to three weeks with continued daily use. Numerous studies have shown an effective dose of HMB to be 3.0 grams per day as CaHMB (calcium HMB) (˜38 mg/kg body weight-day−1). HMB has been tested for safety, showing no side effects in healthy young or old adults. HMB in combination with L-arginine and L-glutamine has also been shown to be safe when supplemented to AIDS and cancer patients.
Recently, HMB free acid, a new delivery form of HMB, has been developed. This new delivery form has been shown to be absorbed quicker and have greater tissue clearance than CaHMB. The new delivery form is described in U.S. Patent Publication Serial No. 20120053240 which is herein incorporated by reference in its entirety.
It has been surprisingly and unexpectedly discovered that HMB modulates both autophagy and lipophagy. The present invention comprises a composition of HMB and methods of use of HMB to result in normalized or modulated autophagic processes. The processes are involved in protecting humans and other animals from the consequences of excessive muscle wasting and atrophy which are common with nutrient deprivation and excesses, as well as activating lipolytic pathways which are necessary for providing energy. It is well established that nutrient excess as well as several diseases (e.g. sepsis, cancer, etc.) are associated with inhibition of lipolysis, and in this regard, HMB modulate the autophagy pathways to mobilize the adipose tissue stores and enhance beta oxidation thus providing energy. HMB's role as an autophagy and lipophagy modulator is depicted in FIG. 1.